Heterochromatin Protein 1 (HP1) is an evolutionary conserved non-histone chromosomal protein that plays a role in the formation of heterochromatin. The main objective of this proposal is to understand the mechanism by which HP1 forms heterochromatin. HP1 is composed of an N- terminal chromo domain that binds methylated lysine 9 of histone H3, a C-terminal chromo shadow domain that acts in dimerization of HP1 and interacts with partner proteins, and an intervening flexible hinge region. We hypothesize that heterochromatin formation results from linking adjacent nucleosomes by HP1, and that the flexible hinge region of HP1 mediates this linkage. Specific Aim I will determine whether mutant forms of HP1 lacking specific functions rescue the lethality associated with an HP1 null. In Drosophila, mutations in the gene encoding HP1 are late larval lethal. Mutant forms of HP1 lacking specific functions (dimerization, interactions with partner proteins, or lacking the hinge region) will be expressed in Drosophila and assayed for their ability to rescue to adulthood. Specific Aim II will determine the role of the HP1 hinge region in the formation of heterochromatin and gene silencing in vivo. Previous studies have demonstrated that the chromo and chromo shadow domains are essential for gene silencing and heterochromatin spreading. Here, we will focus on the role of the hinge region. A mutant form of HP1 lacking the hinge will be expressed in Drosophila and tested for effects on chromatin structure, gene silencing and heterochromatin spreading. Specific Aim III will use in vitro biochemical and biophysical techniques to determine the preferred nucleosome substrate for HP1, and determine the extent to which binding of HP1 results in internucleosomal linkages. Recombinant wild-type and mutant HP1 lacking the hinge region will be purified from E. coli. In collaboration with the Shogren-Knaak laboratory (Iowa State University) we will determine in vitro whether HP1 prefers to interact with mono- or di-nucleosomes by differential equilibrium experiments. Internucleosomal linkages will be evaluated through analytical ultracentrifugation. Relevance: In humans, Heterochromatin Protein 1 (HP1) has been shown to influence cancer progression. For example, levels of HP1 regulate the invasive potential of breast cancer cells. Through the use of a genetically tractable model organism, Drosophila, the proposed studies will greatly advance our knowledge of how HP1 functions in heterochromatin and gene regulation. These findings have implications for the function of HP1 as a metastasis suppressor and will reveal new possibilities for therapeutic approaches.